Dinucleotide repeat in the third intron of the FABP3/MDGI putative tumor suppressor gene.

Abstract

The product of the cardiac fatty acid binding protein (also known as mammary­ derived growth inhibitor: MDGI) has been shown to have modest antiproliferative activity for human breast cancer cells in vitro (Grosse et at., 1991). It has recently been demonstrated that human breast cancer cells transfected with a MDGI expression construct exhibit a reduced proliferation rate and reduced tumorgenicity in nude mice as compared to controls (Huynh et ai., 1995). These results suggest that MDGI has tumor suppressor activity. The FABP3IMDGI gene has been mapped to chromosome 1 at p32p33 (Peeters et ai., 1991; Troxler et at., 1993). Here we describe a polymorphic dinucleotide repeat within the third intron of the FABP3/MDGI gene. This highly informative marker will be extremely useful in genotyping studies of chromosome 1 in the region of FABP3/MDGI. In particular, this polymorphism will be useful in studies of tumor types in which loss of MDGI function might contribute to the tumor phenotype. A AEMBL3 F ABP3 genomic clone was isolated by hybridization with the F ABP3 cDNA. Sequence analysis revealed a (GT) 14 repeat located in the third intron ofthe gene. Unique sequences flanking this repeat were identified and used to design polymerase chain reaction (PCR) primers for amplification of the repeat-containing region. This repeat was found to be highly polymorphic (GenBank Accession no. U40222). Primers were developed flanking this polymorphism [Forward (98F-1): 5'-TGCCTGTCTT AAGGATTTGCTG-3' (Coding strand); Reverse (98F-2): 5'-CACCATTGCGAGCATTCTACCC-3' (Antisense strand)]. PCR amplifications were performed in a total volume of 10 III containing 20 ng genomic DNA, 0.25 11M of each primer, 0.025 11M 32p end-labeled forward primer, 10 mM Tris-HCl pH 8.3, 50 mM KCl , 1.25 mM MgCI2, 25 11M each dNTP, 0.2 U Taq polymerase. Cycling parameters were as follows: 940 C for 30 seconds, 60'C for 30 seconds, and n 'c for 60 seconds for 28 cycles. Samples were electrophoresed in a 6% polyacrylamide gel for 2 hours at 2000Y. The gels were dried and exposed to film (OMAT-AR, Kodak). Allele sizes were determined by comparison to an M13mpl8 DNA sequencing ladder.